INTRODUCTION

Blood cells first appear in the murine embryo in the extraembryonic yolk
sac on embryonic day 7 (E7.0) with only limiting numbers of primitive
erythroid progenitor cells (EryP) detectable
(Palis et al., 1999;
Wong et al., 1986). Over the
next 24 hours, significant expansion in the number of EryP cells
occurs in the yolk sac, and mature erythroblasts expressing embryonic
hemoglobin (βH1) appear in the developing blood islands. While primitive
erythropoiesis constitutes essentially all primitive hematopoiesis in the
early yolk sac (E7.0-8.0), other hematopoietic progenitors are also produced
including limiting numbers of macrophage and megakaryocyte progenitors.
Definitive hematopoietic progenitor cells (progenitors that display the same
culture colony morphology, give rise to the same lineages, and express similar
gene products to myeloerythroid progenitors isolated from the fetal liver and
bone marrow) including high proliferative potential colony forming cells, can
be cultured from the yolk sac at E8.25
(Palis et al., 2001). These
progenitor cells and progenitors arising in the para-aortic splanchnopleura
(P-Sp) region begin to circulate following the onset of cardiovascular
function and migrate to the developing liver by E10. The liver serves as the
predominant site of hematopoiesis until just before birth when the spleen and
bone marrow compartments become seeded with circulating stem cells. While some
of the transcriptional regulation of hematopoietic lineage commitment has been
delineated (Shivdasani and Orkin,
1996), most early markers of the hematopoietic lineage are
co-expressed by the intimately associated angioblasts (endothelial precursors)
and thus specific identification of the hematopoietic precursors has been
elusive (Drake and Fleming,
2000).

The platelet glycoprotein receptor IIb/IIIa
(αIIbβ3; CD41/CD61) is required for normal
platelet hemostatic function (Phillips et
al., 1988). CD41 is a protein composed of two subunits that
interact with CD61 in the presence of calcium to form a functional adhesive
protein receptor. Damage to blood vessels results in the release of a variety
of intracellular mediators as well as initiation of the hemostatic cascade
that in combination activate CD41/CD61 to bind to a variety of proteins
including fibrinogen, fibronectin, von Willebrand factor and vitronectin
(Shattil et al., 1998;
Coller, 1990). Heritable
mutations in either CD41 or CD61 subunits results in a bleeding disorder in
human patients called Glanzmann's thrombasthenia
(Bellucci and Caen, 2002).

CD41/CD61 was once considered to be a platelet specific-receptor complex
and has long been used as a phenotypic marker for the megakaryocyte-platelet
lineage (Phillips et al.,
1988). However, some published information suggests that CD41
expression may not be limited to the platelet lineage
(Berridge et al., 1985;
Fraser et al., 1986;
Tronik-Le Roux et al., 2000;
Tropel et al., 1997). CD41
expression was identified on a subpopulation of
CD34+CD41+CD42- human cord blood cells that
possessed colony forming cells (CFC) and lymphoid and myeloid repopulating
ability (Debili et al., 2001).
In contrast, CD34+CD41+CD42- adult mobilized
peripheral blood cells were enriched in megakaryocyte and erythroid CFC
activity but lacked lymphoid repopulating ability. In murine studies, CD41
co-expression with Kit identified cells in late fetal liver and adult marrow
that possessed a variety of CFC activities and gave rise to T lymphocytes in
thymic organ cultures in vitro (Corbel and
Salaun, 2002). Transplantation of adult marrow
Kit+CD41+ cells suggested that the sorted cells gave
rise to short-term but not long-term hematopoietic repopulation
(Corbel and Salaun, 2002).
Other studies in fetal and adult mice suggested that CD41 was expressed on
most yolk sac progenitor cells at 9.5 days post coitus (dpc) and 10.5 dpc
aorta-gonad-mesonephros (AGM) hematopoietic progenitor cells but on few fetal
liver or adult marrow progenitors
(Mitjavila-Garcia et al.,
2002). In addition, CD41 was expressed on most hematopoietic
progenitor cells derived in vitro from murine embryonic stem (ES) cells
(Mitjavila-Garcia et al.,
2002). Finally, recent data suggest that CD41 expression may serve
as a marker for the onset of definitive hematopoiesis in the murine embryo and
in ES cell-derived hematopoietic cells in vitro
(Mikkola et al., 2002).

In the present work, we have identified CD41 expression on yolk sac cells
coincident with the first appearance of primitive erythroid progenitor cells
(E7.0) and on all yolk sac definitive hematopoietic progenitor cells (E8.25).
Fetal liver and adult marrow hematopoietic progenitor cells were found in both
CD41dim and CD41lo/- populations and both populations
demonstrated long-term repopulating ability in multiple lineages upon
transplantation, however, repopulating ability was enriched in the
CD41lo/- cells. These results demonstrate that CD41 is expressed on
the first hematopoietic progenitor cells of the primitive and definitive
lineages in the murine yolk sac. CD41 expression persists in some
hematopoietic stem and progenitor cells in the fetal liver and adult marrow.
These data provide new insight into the expression of a molecule previously
thought to be lineage restricted and suggest novel roles for the CD41/61
receptor complex.

MATERIALS AND METHODS

Maintenance of mouse colonies

C57BL/6J recipient mice were purchased from Jackson Laboratory (Bar Harbor,
ME). B.6Gpi-1a/BoyJ donor mice were obtained by breeding B.6Gpi-1a (gift of Dr
David Harrison, Jackson Labs, Bar Harbor, ME) with B.6SJL-Pep3B/BoyJ
(purchased from Jackson Labs). The Indiana University School of Medicine
Institutional Animal Care and Use Committee approved the care and use of all
animals in this study.

Hematopoietic cell isolation

Yolk sac

Whole embryo or yolk sac cells were dissected from the decidual tissue of
timed-pregnant dams (E6.5-9.5 gestation embryos) and extensively washed in
phosphate-buffered saline (PBS; Invitrogen, Grand Island, NY) as described
(Yoder and Hiatt, 1997).
Presomitic embryos were staged by the presence of tissue landmarks and later
stages by somite counting (Downs and
Davies, 1993). Dissected yolk sacs were incubated for 60-90
minutes at 37°C in 0.1% collagenase/dispase (Sigma, St. Louis, MO) with
20% fetal bovine serum (FBS, HyClone, Logan, UT) in PBS.

Bone marrow

Lineage depletion of fetal liver and adult bone marrow cells

Removal of lineage antigen-expressing cells from fetal liver cells was
achieved as follows. 0.5-1 μg/ml of rat anti-mouse Gr-1 (granulocytes),
B220 (B lymphocytes), TER-119 (erythroid cells), and CD4/CD8 (T lymphocytes)
monoclonal antibodies (PharMingen, San Diego, CA) were added to the fetal
liver cell suspension for a 20 minute incubation on ice. The stained cell
populations were pelleted (300 g for 8 minutes), washed with
buffer, repelleted and resuspended in buffer. Goat anti-rat IgG magnetic
microbeads (Miltenyi Biotec, Auburn, CA) were added for a 20 minute incubation
on ice, and the lineage-depleted cell population was obtained using a magnetic
separation device as directed by the manufacturer (Miltenyi Biotec). This
procedure was repeated for the adult bone marrow cells, with the exception
that purified and fluorescein isothiocyanate (FITC)conjugated rat anti-mouse
Mac1 was also used. Lineage-depleted populations of cells were restained with
FITC conjugates of the same antibodies and analyzed for purity using a
FACSvantage instrument (Becton Dickinson).

Hematopoietic progenitor cell assay

Sorted cells were plated (500-1000 cells/dish) in triplicate in 0.9%
methylcellulose cultures as previously described
(Yoder et al., 1995). Briefly,
the cells were suspended in 1% methylcellulose, 30% fetal calf serum,
10-5 mol/l 2-mercaptoethanol (Sigma), 2 mmol/l glutamine
(Invitrogen), 4 U Epo, 200 U IL-3, and 100 ng SCF. The cells were incubated at
37°C in 5% CO2 in air and groups of more than 50 cells were
scored as colonies (CFU). The early appearing CFU-Meg (<E8.25 plated cells)
were identified as clusters of 3 or more large refractile cells on day 3 of
culture. These cells were plucked, applied to glass slides, and stained for
expression of acetylcholine esterase. These early CFU-Meg were no longer
detectable by day 7 of culture (when the definitive progenitors were
scored).

Whole-mount immunolabeling for confocal microscopy

C57BL/6J mice were killed by cervical dislocation on days 6.5 to 8.5 of
development (day 0.5, morning of vaginal plug). The embryos were dissected and
washed three times in PBS, fixed 10 minutes in cold acetone then rinsed three
more times in PBS. Embryos were blocked in PBS containing 3% blotting grade
non-fat dry milk (Bio-Rad Laboratories, Hercules, CA) and varying amounts of
Triton X-100 (0.0125% for day 7 embryos and 0.025% for day 8 embryos) for 1
hour. Directly conjugated primary antibodies were then added to a final
concentration of 5 μg/ml for 12 to 18 hours at 4°C. TER119, Flk-1 and
CD41 purified antibodies were labeled with Rhodamine Red-X protein labeling
kit (TER119), Alexa Fluor 488 monoclonal labeling kit (Flk-1) or Alexa Fluor
647 monoclonal labeling kit (CD41) (Molecular Probes, Eugene, OR). Similarly
labeled isotype control antibodies produced no specific staining (not shown).
Embryos and yolk sacs were mounted in 70% glycerol/PBS either intact or
embryonic dorsal side up following open dissection according to a previously
published method (Drake and Fleming,
2000).

Fibrinogen staining protocol

Single cell suspensions of YS cells were stained with 1 μl CD61 PE and
sorted into CD61+ and CD61- populations using the
FACStar instrument. Recovered cells were pretreated with 5 μl of 0.1 M DTT
(Invitrogen, Grand Island, NY) for 5 minutes followed by addition of 0.5 μl
of fibrinogen conjugated to Oregon Green (stock solution of 1.5 mg/ml in 0.1 M
sodium bicarbonate, pH 8.3; Molecular Probes, Eugene, OR). The cells were
incubated for 30 minutes at 37°C. To the fibrinogen and cell suspension, 1μ
l CD41 conjugated to Alexa 647 was added for 30 minutes at 37°C.
Following the incubation, cells were pelleted, washed and resuspended in IMDM
with 10% FBS, 1% P/S, 2 mM L-glutamine for cell analysis on the FACSVantage
instrument. In some experiments, 1-8 μl of a anti-mouse CD41 blocking mAb
(1B5F(ab)′2, 1.6 mg/ml) was added to one aliquot and 1-8μ
l of a rat isotype control (99-C7-B3, 1.8 mg/ml) was added (both
antibodies were generously provided by Dr Barry Coller, Rockefeller
University, New York) to a second aliquot of cells for 30 minutes at 37°C
prior to addition of fibrinogen.

RESULTS

CD41 cell surface expression

CD41-expressing cells were first detectable in E7.0
(Fig. 1B) cells isolated from
the entire developing embryo. Both the percentage and intensity of staining
increased by E9.5 in the yolk sac (Fig.
1D). Several different yolk sac cell populations could be
discriminated by the intensity of CD41 expression at E9.5 with
CD41bright, CD41dim and CD41lo/- patterns
(Fig. 1D). The intensity and
percentage of cells expressing CD41 diminished in the fetal liver and bone
marrow populations compared with that in yolk sac cell populations
(Fig. 1H,I). CD34 is a
sialomucin highly expressed on endothelial and some hematopoietic cells
(Krause et al., 1996). CD34
and CD41 co-expression was evident in whole embryo (E7.0;
Fig. 1B), yolk sac (E8.0-E10.0;
Fig. 1C,D,F), embryo proper
(E9.5-10.0; Fig. 1E,G), fetal
liver (E12.5; Fig. 1H) and
adult bone marrow cells (Fig.
1I) with the highest percentage of co-expressing cells present in
the yolk sac (Fig. 1D,F). Of
interest, the percentage and level of intensity of CD41 expression was less in
cells isolated from the embryo proper (Fig.
1E,G) than in similarly staged and paired yolk sac samples
(Fig. 1D,F).

Colony forming potential of CD41-expressing cells in the early yolk
sac

CD41dim and CD41bright expressing cells were
appearing in the yolk sac at times coincident with the appearance of
EryP and definitive hematopoietic progenitor cells, respectively,
and suggested that these progenitors may be expressing CD41. In fact,
CD41dim cells arising at E7.0 in the yolk sac possessed all of the
EryP-CFC activity at this stage
(Table 1). The
EryP-CFC activity remained essentially restricted to the
CD41dim population through E8.5
(Table 1) after which time
these progenitors became undetectable (data not shown). All of the CFU-Mac
found at E8.0 were also present in cultures initiated with CD41dim
cells. Even if 20-fold more cells were plated, no CFU-Mac were present in the
CD41lo/- population (data not shown). Similarly, all of the CFU-Meg
detectable at E7.5 were present in the CD41dim fraction. These data
indicate that CD41dim cells in the early yolk sac contain all of
the macrophage, megakaryocytic and primitive erythroid cell activity as these
cells first emerge as detectable progenitor cells. Thus, CD41dim
expression serves as a marker for the onset of megakaryopoiesis, macrophage
CFC, and primitive erythroid progenitor cell emergence in the murine yolk
sac.

Definitive hematopoietic progenitor cells are first detectable in the yolk
sac at E8.25 (Palis et al.,
1999). We examined E8.0-8.25 yolk sac cells for expression of CD41
and identified a new population of CD41bright cells in addition to
CD41dim and CD41lo/- cells
(Fig. 1C). Plating of these
cells in progenitor assays revealed no CFC growth from plated
CD41lo/- cells, however, nearly all definitive progenitor cells
were present in the CD41bright population whereas the
CD41dim cells retained the EryP-CFC and low numbers of
definitive progenitors (Table
1). As expected, βH1 and β globin major mRNA were
expressed in the EryP-CFC-derived erythroblasts present in the
cultures of sorted CD41dim cells
(Fig. 2). Proof that the
CD41bright population gave rise to definitive erythroid cells was
confirmed by the presence of β globin major but not βH1 mRNA in the
erythroid cells from isolated BFUEs (Fig.
2). In summary, these results indicate that essentially all
definitive progenitor cell activity is present in the CD41bright
population of E8.25 yolk sac cells and that nearly all CD41dim
cells persisting at this stage continue to possess EryP-CFC
activity. Thus CD41 expression serves as a marker for the onset of definitive
as well as primitive hematopoiesis in the murine yolk sac.

Colony morphology and analysis of hemoglobin mRNA expression.
Photomicrograph of an E8.5 yolk sac primitive erythroid cells (EryP) and a
definitive erythroid progenitor cell (BFU-E). Magnification ×100. Panels
to the right of the photomicrographs depict the results of RT-PCR analysis of
plucked erythroid colonies and reveal that EryP contain mRNA forβ
H1 (embryonic hemoglobin) and β globin major (adult hemoglobin)
while BFU-E express mRNA for only β globin major. Negative control
samples were tested using no reverse transcriptase.

Spatial and temporal analysis of the emergence of CD41-expressing
cells

Expression of Flk-1 and CD41 in the gastrulating murine embryo. (A) E7.0
(mid-streak) embryo reveals Flk-1-expressing putative mesoderm cells migrating
from the level of the embryo proper to the more proximal region of the egg
cylinder (white box indicates region depicted in B and C). (B) Flk-1 staining
only. (C) Emergence of CD41dim expression in the E7.0 yolk sac
(arrowheads). (D) E7.25 (late-streak stage) embryo expresses both Flk-1 (E)
and CD41dim (F) cells with the CD41dim cells forming a
band of cells in the most proximal portion of the yolk sac while
Flk-1+ cells extend toward the distal leading edge of the yolk sac
as it approaches the embryo proper. (G) A composite higher magnification image
of the proximal yolk sac (in orthogonal view) of an E7.25 embryo. The
unlabeled visceral endoderm (endo) overlies Flk-1+ (H) and
CD41dim (I) expressing cells. The arrowheads (G-I) reflect cells in
transition from Flk-1+ CD41lo/- (green) to
Flk-1+ CD41dim (cyan) to Flk-1dim
CD41dim (blue). (J) E7.5 (neural plate stage) embryos reveal a
wider distribution of Flk-1-expressing cells with appearance of numerous
capillary-like structures. The band of CD41dim-expressing cells has
expanded but remains centered at the proximal end of the yolk sac. Arrows
indicate contaminating maternal red blood cells expressing high levels of the
erythroid marker, TER119 (small bright red cells also present in D and
Fig. 4B). (K) Reconfiguration
of the neural plate stage embryo into a planar fashion by dividing the embryo
from the most proximal to distal, results in a clear view of the continuity of
the CD41dim-expressing cells for the full circumference of the yolk
sac. (L) Flk-1 expression alone in the proximal region of the yolk sac. Some
of the cells are Flk-1bright and many are Flk-1dim and
these cells correspond to the same cells (M) that are CD41dim; the
band of CD41-expressing cells is 5-10 cells in width and 1 cell thick. Flk-1
highly expressing cells overlie the band of
Flk-1dimCD41dim cells but an orthogonal view (N) reveals
that the Flk-1bright cells (asterisks) are interposed between the
band of CD41dim cells and the outer endoderm layer (endo). Scale
bar: 100 μm (A-F,J-M) and 33 μm (G-I,N).

CD41dim fluorescence was first detected at the mid-streak stage
(E7.0) in a narrow ring of Flk-1+ cells at the proximal end
(farthest from the embryo proper) of the yolk sac
(Fig. 3A-C). By the late-streak
stage, CD41+ cells form a uniform band 5-10 cell diameters in width
that remains centered around the proximal portion of the yolk sac overlying
the upper exocoelomic cavity (Fig.
3D-F). Higher magnification of the yolk sac at this stage
permitted visualization of a progression of cellular immunophenotypes from
Flk-1+CD41lo/- to Flk-1+CD41dim to
Flk-1lo/-CD41dim
(Fig. 3G-I). The band of
CD41dim-expressing cells remained constant in size (5-10 cell
diameters wide and 1-2 cells thick) and intensity through the neural plate
stage of development (Fig.
3J-M). Examination of dissected neural plate stage embryos
revealed that the band of CD41dim cells was contiguous around the
entire circumference of the yolk sac (Fig.
3K-M). Examination of a neural plate stage yolk sac in orthogonal
section revealed that the CD41+ cells were not circumscribed by the
Flk-1+ developing endothelium. These Flk-1+ nascent
endothelial cells (likely angioblasts) are situated between the visceral
endoderm and the CD41+ primitive erythroid elements
(Fig. 3N).

Emergence of the Ery P lineage and the definitive
progenitors

Expression of the erythroid lineage marker TER119 in a subset of the
CD41dim cells began soon after the onset of blood island
vasculogenesis (Fig. 4A-C).
Between E8.0 and E8.25 there was a significant increase in the number of
TER119+ cells and the level of TER119 expression
(Fig. 4A,D). Cells with the
highest level of TER119 expression had diminished levels of CD41 expression
(below dim), thus a reciprocal relationship appeared to exist between the
level of TER119 expression and the level of CD41 expression in the
CD41dim population (Fig.
4E-I). When CD41dim and TER119 co-expressing cells
(Fig. 4I) were sorted, the
frequency of EryP in E8.5 yolk sac
CD41dimTER119- cells was 83/2000 cells plated compared
to 41/2000 cells plated for the more mature
CD41lo/-TER119+ cells (n=2). These observations
suggest that maturing EryP and emerging primitive erythroblasts
downregulate CD41 expression during differentiation
(Fig. 4E-I). However, the level
of TER119 expression in the primitive erythroid lineage never attained that
displayed by definitive erythroid cells (e.g. contaminating maternal
erythrocytes in Fig. 3J).

Emergence of TER119-expressing erythroid cells and CD41bright
cells in the E8.0-8.25 embryo. (A) A portion of the emerging blood island in
an E8.0 embryo (1-somite-pair stage) with the first TER119-expressing cells.
(B) Extent of TER119 expression in cells (note the presence of
TER119bright maternal erythrocytes). (C) Emergence of a new
population of CD41bright cells among the CD41dim cells.
(D) E8.25 (4 somite pairs) embryo that has been rendered planar with the
anterior (ant) and posterior (post) regions, allantoic stalk (all), and
somites (s) identified. Massive expansion in the TER119-expressing cells and
extensive vasculogenesis with a network of capillary-like structures is
evident as is the emergence of CD41bright cell clusters
(asterisks). White box indicates region depicted in E-H. (E) High
magnification composite image of a blood island containing TER119- (red),
Flk-1- (green), and CD41- (blue) expressing cells. Isolated channels are shown
in F-H. A red arrow in E-H identifies a cell expressing TER119 (red) but not
CD41 (blue). A blue arrow (EH) indicates a cell expressing only CD41 (blue). A
yellow arrow indicates a cell that is expressing TER119 (red) and CD41 (blue).
Of interest the CD41bright cell in panel G (asterisk) also
expresses Flk-1 (E,H). Scale bar: 100 μm (A-D) 33 μm (E-H). (I) A
representative dot plot of sorted E8.25 whole embryos with quadrants
color-coded to match arrows in E-H.

The onset of TER119 expression in the CD41dim population between
E8.0 and early somite stages (E8.25) was accompanied by the emergence of a
novel CD41bright population of cells
(Fig. 4). These
CD41bright cells were always TER119- and seemed to be
intimately associated with the endothelium of the developing vasculature
(Fig. 4C,G). Some of these
CD41bright cells coexpressed Flk-1
(Fig. 4E-H asterisk). The
CD41bright cells were found in clusters of 10-20 cells at the
borders between the blood island and the capillary bed leading to the embryo
proper (Fig. 4D asterisks).
These cells appear to expand in the yolk sac at the time of appearance of the
first definitive progenitor cells (E8.25) and since essentially all definitive
progenitors are CD41bright
(Table 1) at least some of the
clustered CD41bright cells must be definitive progenitors.

CFC potential of CD41+ cells in the late yolk sac, PSp,
fetal liver and adult marrow

Yolk sac, P-Sp, fetal liver and adult marrow
Kit+CD34+ cells are enriched for hematopoietic
progenitor cell activity (Ito et al.,
2000). We examined Kit+CD34+ cells in the
E9.5 yolk sac, E12.5 fetal liver and adult bone marrow for expression of CD41
(Fig. 5). Nearly all
Kit+CD34+ cells in the E9.5 yolk sac express CD41
(Fig. 5A). CD41 expression was
present on a high percentage of Kit+CD34+ cells in the
E12.5 fetal liver (Fig. 5E) but
expression was restricted to a small percentage of adult bone marrow cells
(Fig. 5F). Since adult bone
marrow hematopoietic stem cells are enriched in cells that fail to express
CD34, we also examined Kit+CD34- cells for CD41
expression and less than 1% of these cells expressed CD41 (data not shown).
CD41 expression was also examined in E9.5 Kit+CD34+ P-Sp
cells and the majority of cells (68±12%) were CD41+.

Co-expression of CD41 and CD61 with fibrinogen binding activity in
yolk sac cells

We examined E9.5 yolk sac cells for co-expression of CD41 and CD61 as these
proteins interact to bind fibrinogen in mature platelets. CD41 detected on the
cell surface of E9.5 yolk sac cells co-localized with CD61, though not all
CD61 molecules co-localized with CD41 (Fig.
8). When we isolated CD61-expressing E9.5 yolk sac cells
(Fig. 8) and then examined
cells for CD41 expression and fibrinogen binding, we observed significant
co-localization of the two fluorochromes
(Fig. 8). In preliminary
studies, addition of monoclonal antibody (1B5F(ab)′2) to the
yolk sac cells before exposure to fibrinogen blocked 50% of the fibrinogen
binding whereas a non-blocking control antibody had no effect (data not
shown). These data indicate that some of the non-megakaryocytic hematopoietic
progenitor cells in the E9.5 yolk sac possess the capacity to bind soluble
fibrinogen in vitro.

DISCUSSION

We report the first strategy for the isolation of both primitive and
definitive hematopoietic progenitor cells in the murine embryo. CD41
expression marks the first primitive erythroid and essentially all definitive
hematopoietic progenitor cells in the yolk sac at the times these progenitors
are first detectable with in vitro clonogenic assays. As development proceeds,
CD41 expression is lost on some fetal liver and most adult marrow
hematopoietic progenitor cells. However, CD41 expression is retained on some
long-term repopulating hematopoietic stem cells that can reconstitute for at
least 6 months following transplantation.

The transient nature, unique morphology and gene expression, and restricted
cell types of the primitive erythroid lineage suggest that it is distinct from
the definitive erythroid lineage. Support for the separation of primitive and
definitive erythropoiesis is also provided by gene-targeting experiments where
deletion of certain genes (e.g. Runx1, Gata2) disrupts definitive
erythropoiesis with little change in primitive erythropoiesis
(Shivdasani and Orkin, 1996).
The early appearing macrophage progenitors identified in the present studies
(E8.0) are absent in Runx1 null embryos and are therefore probably
related to the definitive lineage (Lacaud
et al., 2002). Whether the early appearing megakaryocyte
progenitors identified in the present studies are more related to the
definitive or primitive lineage remains to be determined, but these
progenitors are reported to display features that distinguish them from later
appearing definitive megakaryocyte progenitors
(Xu et al., 2001). Studies
performed with murine embryonic stem (ES) cells suggest that primitive and
definitive hematopoietic progenitor cells emerge from a common
Flk-1+ precursor cell called the hemangioblast
(Kennedy et al., 1997).
Expression of Flk-1 is first evident in the murine embryo in proximal lateral
mesoderm during gastrulation and these mesoderm cells give rise to both the
hematopoietic and endothelial lineages in the yolk sac
(Kataoka et al., 1997;
Nishikawa, 1997). While Flk-1
has been used to isolate primitive and definitive progenitor cells from ES
cell-derived hematopoietic cultures, Flk-1 has not been used to purify
EryP or early appearing CFU-Mac and megakaryocytic progenitors from
the early yolk sac. We have identified CD41 expression in the yolk sac in
Flk-1+ cells and have demonstrated that the
CD41dim-expressing yolk sac cells at E7.0 constitute the entire
population of in vitro clonable EryP-CFC. Thus, CD41 expression
serves as a marker for both the onset of hematopoiesis in general (early
macrophage and megakaryocytic progenitors), and specifically for primitive
erythroid progenitor cell emergence.

CD41 expression at a high level in E8.25 yolk sac cells identifies
essentially all of the definitive hematopoietic progenitor cells. Flk-1 and
vascular endothelial cadherin have previously been utilized to isolate
primitive and definitive hematopoietic cells in vitro from ES cell-derived
hematopoietic cultures but this combination has not been examined as a tool
for isolating primitive and definitive progenitors in the early yolk sac
(Kabrun et al., 1997;
Nishikawa et al., 1998). The
core-binding factor Runx1 is expressed in endoderm, mesoderm and primitive
erythroblasts in the yolk sac (North et
al., 1999). It remains to be determined whether expression of this
transcription factor will identify EryP or CFU-Mac in the early
yolk sac. Thus, CD41 is a unique marker for both primitive and definitive
hematopoietic progenitor cells in the earliest phases of murine development.
These observations both support and extend the recent reports of CD41
expression on hematopoieitc progenitor cells from human and murine fetal and
newborn subjects and the report that CD41 serves as a marker for the onset of
definitive hematopoiesis (Corbel and
Salaun, 2002; Debili et al.,
2001; Mikkola et al.,
2002; Mitjavila-Garcia et al.,
2002).

While essentially all the hematopoietic progenitor cells in the yolk sac
expressed CD41, CD41 expression diminished in progenitors present in the P-Sp,
fetal liver and adult bone marrow compartments. We have summarized the
immunophenotype of these progenitors in
Fig. 9. E9.0 yolk sac
repopulating stem cells are enriched in cells expressing CD34 and Kit and
nearly all of these cells expressed CD41 in the present studies
(Yoder et al., 1997b). CD34
and Kit are expressed on hematopoietic stem and progenitor cells in the AGM,
fetal liver and adult marrow (Morel et
al., 1996; Sanchez et al.,
1996). Of interest, fetal liver stem cell activity is enriched in
cells expressing CD34 and Kit while adult marrow stem cell repopulating
activity is highest in cells expressing Kit but not CD34
(Ema and Nakauchi, 2000;
Ito et al., 2000;
Zeigler et al., 1994). We
observed that transplantation of lethally irradiated adult mice with sorted
fetal liver and adult bone marrow cells resulted in very low but persistent
long-term chimerism of the peripheral blood of recipient mice with donor cells
expressing CD41. However, the highest levels of donor cell chimerism achieved
in the competitive repopulation experiments resulted from transplantation of
Kit+CD34+ fetal liver and
Kit+CD34- adult marrow cells that did not express
detectable cell surface levels of CD41. Further studies to determine whether
CD41 is expressed on stem cells derived from the AGM, the site of development
of the first stem cells that engraft in adult mice are warranted.

Model of CD41 expression on various hematopoietic cell subsets. The results
of the present studies lead us to predict a model whereby the primitive and
definitive erythroid progenitor cells can be discriminated by the level of
CD41 expression. Likewise, CD41 expression varies on long-term repopulating
bone marrow stem cell subsets with the cells possessing the greatest
repopulating ability expressing neither CD34 or CD41 (yellow cell) on the cell
surface.

CD41/CD61 interactions on the surface of platelets plays an important role
in the adhesion, spreading and aggregation of platelets in areas of vascular
injury via interactions with a variety of extracellular matrix and plasma
proteins. We have identified fibrinogen binding to CD41- and CD61-expressing
hematopoietic progenitor cells in E9.5 yolk sac cells. Plating of cells that
bind fibrinogen alone does not isolate or enrich for hematopoietic progenitor
cells, however, plating cells that express CD41 or CD61 and fibrinogen
enriches for yolk sac progenitors. These results further support the growing
appreciation that the CD41/CD61 complex is not restricted to the platelet
lineage.

The role CD41 is playing in the primitive and definitive progenitor cells
is unknown. One obvious role the CD41/61 receptor complex may play is in the
interaction of the progenitor cells with extracellular matrix molecules in the
yolk sac. The specific adhesion interactions between the earliest
hematopoietic and endothelial cells that are required for proper formation of
yolk sac blood islands remain unknown. Disruption of several integrin and
extracellular matrix molecule genes results in embryonic lethality due to
aberrant blood island and yolk sac vascular development (with or without
defects in hematopoiesis) (Francis et al.,
2002). Thus, CD41 may play a role in hematopoietic-endothelial
interactions important for early blood island organogenesis. However, mice
deficient in CD41 display no obvious defect in yolk sac, fetal liver or adult
marrow hematopoiesis other than a platelet adhesion abnormality similar to
that seen in human subjects with Glanzmann's thrombasthenia
(Tronik-Le Roux et al., 2000).
Thus, further studies will be required to define the role of CD41 in
hematopoietic stem and progenitor function during murine embryogenesis and
throughout ontogeny.

Similar articles

Other journals from The Company of Biologists

Although sad to be saying goodbye to Ottoline Leyser and Geraldine Seydoux as they leave our Editorial Board, we are very pleased to introduce two new editors, Yka Helariutta and Susan Strome. Olivier Pourquié has also announced that he will also be stepping down next year, and so we invite the Development community to help us make the right choice for our next Editor in Chief.

“It's good advice that you collaborate – you need to have so many aspects to what you are doing and you can't possibly be an expert in every one”

Jenny Nichols, winner of the 2017 BSDB Cheryll Tickle Medal, talks about the importance of collaboration throughout her career investigating pluripotency in the mammalian embryo and what playing a musical instrument has in common with research.

Organised by Paola Arlotta, Ali Brivanlou, Olivier Pourquié and Jason Spence, the third meeting in our highly successful series of events focussing on human developmental biology will be held at Wotton House in Surrey, UK, on 23 - 26 September 2018. Mark it on your calendar now!

The authors of a Research article on how PLCζ-null mice can still be fertile even in the absence of the Ca2+ oscillations that induce egg activation chat to the Node about their research, looking positively on bad luck and bringing science to life for children.

Dorit Hockman from the University of Oxford studies the development and evolution of the neural crest by investigating its development in lampreys. A Travelling Fellowship from Development gave Dorit the opportunity to visit Marianne Bronner’s laboratory at the California Institute of Technology over the lamprey breeding season, where she learned how to track neural crest cells as they migrated into the branchial arches. Read her story here.

Where could your research take you? The deadline for the current round of applications for a Development Travelling Fellowship is 31 August. Find out more here.